Protein C (PC) is activated on the surface of endothelial cells by the thrombin-thrombomodulin complex and the generated activated PC (APC) inhibits blood coagulation by down regulating prothrombin activation in a negative feedback loop. In addition to its role as an anticoagulant, APC has major anti-inflammatory effects that led to its recent approval for treatment of patients with severe sepsis. The molecular basis for APC's important protective effects is incompletely understood. This application is based upon our recent identification of protease-activated receptor 1 (PAR1) and endothelial cell PC receptor (EPCR) as part of a novel APC signaling pathway in endothelial cells. The general hypothesis is that PARl-dependent signaling of the PC system in endothelial cells limits inflammatory responses and reduces lethality in sepsis. PAR1 can be efficiently activated by thrombin and Aim#1 is to define how autocrine PAR1 signaling by APC is linked to PC activation on the cell surface. Experiments will analyze whether activation of EPCR-bound PC by thrombin-thrombomodulin leads to enhanced APC signaling and what the role of membrane compartmentalization of PC pathway components and PAR1 is. Aim#2 is to define the role of PAR1, EPCR, and thrombomodulin in APC-dependent effects on gene expression, including the down regulation of genes involved in inflammatory and apoptotic pathways in activated endothelial cells. Micro array analysis will identify downstream targets of protective PC pathway signaling and adenovirus-mediated expression of mutant EPCR and thrombomodulin will define the roles of these cofactors. Aim#3 is to demonstrate an in vivo role of PAR1 activation by the PC pathway. Transgenic mice will be generated with endothelial cell-specific expression of either wild type PAR1 or a variant PAR1 that can be activated by APC but not thrombin. Effects of APC administration on inflammatory responses and lethality in a model of septicemia will be analyzed in the mice and the results are expected to define the role of endothelial PAR1 in systemic inflammation and in mediating protective effects of APC.